Nucleic Acid Detection Methods Research Articles

Methods for enrichment and nucleic acid detection of SARS-CoV-2 in sewage

Since the outbreak of COVID-19, SARS-CoV-2 surveillance in sewage based on sewage epidemiology has been established in many countries around the world, while there is a lack of a standard detection method for SARS-CoV-2 in sewage in China. This standard provides three methods for the enrichment of SARS-CoV-2 in sewage, namely polyethylene glycol precipitation, aluminum salt coagulation precipitation and centrifugal ultrafiltration, which are suitable for the enrichment and nucleic acid detection of SARS-CoV-2 in domestic sewage and sewage from medical institutions. This standard is of great significance for standardizing the SARS-CoV-2 detection in sewage and ensuring the scientific and effective detection of SARS-CoV-2 in sewage.

Development of Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of Human Norovirus Major Capsid Protein.

Human Norwalk viruses (HuNoVs), the most common etiological agents of acute gastroenteritis, are genetically diverse RNA viruses that frequently cause mass food poisoning internationally. Although nucleic acid detection methods, such as reverse transcription–quantitative polymerase chain reaction (RT-qPCR), are the gold standard for the diagnosis of norovirus infection, alternative methods are needed for the specific and sensitive viral protein detection for rapid diagnosis and surveillance. In this study, we developed a robust and high-throughput targeted proteomic assay workflow to directly detect the VP1 major capsid protein of HuNoVs. A parallel reaction monitoring (PRM) assay using a high-resolution mass spectrometer was used to detect representative peptides derived from VP1 in six different HuNoV genotypes. An optimized protocol using synthesized heavy isotope-labeled peptides as internal standards was also used to simultaneously genotype and quantify the VP1 protein in human stool specimens. This method is expected to become a new tool for studying the molecular epidemiology of HuNoV and to shed new light on targeted proteomics in clinical practice.

A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction

Existing detection methods for pathogen nucleic acid detection, such as polymerase chain reaction (PCR), are complicated and expensive to perform. Here, we report a simple and versatile strategy for highly sensitive detection of pathogen nucleic acid based on toehold-mediated strand displacement initiated primer exchange amplification (t-PER). In the presence of the target, the blocked hairpin substrate is released by toehold-mediated strand displacement, which triggers the primer exchange reaction amplification. Then, multiple long tandem-repeat single-strands generated by PER open the molecular beacon to recover the fluorescence signal. The t-PER protocol also successfully directly detected human papilloma virus from clinical cervical swab samples, with consistent results compared to real time-polymerase chain reaction (RT-PCR). Moreover, the versatility and clinical feasibility of this method was further confirmed by measuring Epstein-Barr virus, hepatitis B virus, and Ureaplasma urealyticum from different clinical samples (serum samples and urine samples). This simple platform enabled specific and sensitive detection of pathogen nucleic acid in a format that might hold great potential for point-of-care infection diagnosis.

Rapid and Ultrasensitive Detection of Methicillin-Resistant Staphylococcus aureus Based on CRISPR-Cas12a Combined With Recombinase-Aided Amplification.

Staphylococcus aureus is one of the main pathogens causing hospital and community-acquired infections, in particular, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) cause a higher mortality rate than those caused by methicillin-sensitive strains, which poses a serious global public health problem. Therefore, rapid and ultrasensitive detection of patients with clinical MRSA infection and timely control of infection are essential. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) based on nucleic acid detection methods are well-known for its high specificity and sensitivity and programmability. Here, we successfully proposed a method based on CRISPR-Cas12a combined with recombinase-aided amplification (RAA) through fluorescent readout to achieve accurate identification and highly sensitive detection of MRSA in clinical samples. Results showed that the limit of detection (LoD) of the RAA-Cas12a method could reach 10 copies/μl at 60 min of reaction. Specificity tests showed that the method could distinguish MRSA from clinically common bacteria. The results of RAA-Cas12a were consistent with that of antimicrobial susceptibility tests (AST) and polymerase chain reaction (PCR) in 83 clinical samples. These results indicated that the detection method based on RAA-Cas12a has high sensitivity and specificity, and provides important value for rapid detection of MRSA.

A Highly Sensitive and Specific Detection Method for Mycobacterium tuberculosis Fluoroquinolone Resistance Mutations Utilizing the CRISPR-Cas13a System.

ObjectivesCRISPR-Cas13a system-based nucleic acid detection methods are reported to have rapid and sensitive DNA detection. However, the screening strategy for crRNAs that enables CRISPR-Cas13a single-base resolution DNA detection of human pathogens remains unclear.MethodsA combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was proposed.ResultsA set of crRNAs was found to enable the CRISPR-Cas13 system to dramatically distinguish fluroquinolone resistance mutations in clinically isolated Mycobacterium tuberculosis strains from the highly homologous wild type, with a signal ratio ranging from 8.29 to 38.22 in different mutation sites. For the evaluation of clinical performance using genomic DNA from clinically isolated M. tuberculosis, the specificity and sensitivity were 100 and 91.4%, respectively, compared with culture-based phenotypic assays.ConclusionThese results demonstrated that the CRISPR-Cas13a system has potential for use in single nucleotide polymorphism (SNP) detection after tuning crRNAs. We believe this crRNA screening strategy will be used extensively for early drug resistance monitoring and guidance for clinical treatment.

An ultrafast ratiometric electrochemical biosensor based on potential-assisted hybridization for nucleic acids detection

The development of rapid nucleic acid detection methods, which are promising the diagnostic standard of the infectious disease, could expand more options for disease traceability and controllability. Nucleic acid hybridization-based biosensing techniques still encounter limitations in meeting the requirements for rapid detection. Therefore, we proposed a potential-assisted ratiometric electrochemical biosensor for rapid and accurate target DNA detection. This dual-signal analysis system actuated an enzyme-free detection process. The application of an external electric field accelerated molecular dynamics, resulting in highly efficient collision chances. This hybridization process was thus improved from hours to minutes compared with passive hybridization approach. The biosensor not only had a high sensitivity with the detection limit of 12 fM, but also features a robust capability in identifying single-base mismatch. Moreover, the biosensor realized sensitive detection of target DNA in complex biological environments. Overall, this sensing strategy exhibits a promising potential for application in point-of-care testing.

Fluorescence microarrays for enzyme-free DNA detection based on web hybrid chain reaction

The diagnosis of pathogenic microorganism infection mainly relies on molecular detection of nucleic acids or proteins, and the nucleic acid detection is the gold standard for the pathogen detection. The viral genome mutates quickly, and the genome of the host cell usually binds to multiple sites in the viral genome, leading to patient infections. Therefore, rapid nucleic acid detection methods with high specificity are essential for controlling the spread of viruses and maintaining human health. In this study, Cy3-labeled DNA probes were obtained based on a convenient and enzyme-free web hybridization chain reaction (wHCR) and used for DNA diagnosis of hepatitis B virus (HBV) in an isothermal platform. In this microarray platform, the target DNA is directly captured on the microarray and identified by the aggregated DNA probes to amplify the fluorescence signal. After fluorescence scanning analysis, the detection limit of HBV DNA fragments on this DNA microarray is 84 fM. In addition, this method not only specifically distinguishes single-base mismatched sequences, but also obtains the quantitative detection of HBV DNA in serum samples. Compared with the enzymatic amplification reaction in which the target nucleic acids are used as the amplification template, this method effectively avoids cross-contamination. Furthermore, compared to enzymatic reactions, the requirements for reaction conditions such as temperature and pH are relatively lenient. This method is user-friendly and suitable for molecular detection of infectious diseases under limited resource conditions.

Application of Nanopore Sequencing in the Detection of Foodborne Microorganisms.

Foodborne pathogens have become the subject of intense interest because of their high incidence and mortality worldwide. In the past few decades, people have developed many methods to solve this challenge. At present, methods such as traditional microbial culture methods, nucleic acid or protein-based pathogen detection methods, and whole-genome analysis are widely used in the detection of pathogenic microorganisms in food. However, these methods are limited by time-consuming, cumbersome operations or high costs. The development of nanopore sequencing technology offers the possibility to address these shortcomings. Nanopore sequencing, a third-generation technology, has the advantages of simple operation, high sensitivity, real-time sequencing, and low turnaround time. It can be widely used in the rapid detection and serotyping of foodborne pathogens. This review article discusses foodborne diseases, the principle of nanopore sequencing technology, the application of nanopore sequencing technology in foodborne pathogens detection, as well as its development prospects.

Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a simple proof of principle workflow using stabilized reagents and cell phone camera optical readout, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing.

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.

Aptamers as promising nanotheranostic tools in the COVID-19 pandemic era.

The emergence of SARS‐COV‐2, the causative agent of new coronavirus disease (COVID‐19) has become a pandemic threat. Early and precise detection of the virus is vital for effective diagnosis and treatment. Various testing kits and assays, including nucleic acid detection methods, antigen tests, serological tests, and enzyme‐linked immunosorbent assay (ELISA), have been implemented or are being explored to detect the virus and/or characterize cellular and antibody responses to the infection. However, these approaches have inherent drawbacks such as nonspecificity, high cost, are characterized by long turnaround times for test results, and can be labor‐intensive. Also, the circulating SARS‐COV‐2 variant of concerns, reduced antibody sensitivity and/or neutralization, and possible antibody‐dependent enhancement (ADE) have warranted the search for alternative potent therapeutics. Aptamers, which are single‐stranded oligonucleotides, generated artificially by SELEX (Evolution of Ligands by Exponential Enrichment) may offer the capacity to generate high‐affinity neutralizers and/or bioprobes for monitoring relevant SARS‐COV‐2 and COVID‐19 biomarkers. This article reviews and discusses the prospects of implementing aptamers for rapid point‐of‐care detection and treatment of SARS‐COV‐2. We highlight other SARS‐COV‐2 targets (N protein, spike protein stem‐helix), SELEX augmented with competition assays and in silico technologies for rapid discovery and isolation of theranostic aptamers against COVID‐19 and future pandemics. It further provides an overview on site‐specific bioconjugation approaches, customizable molecular scaffolding strategies, and nanotechnology platforms to engineer these aptamers into ultrapotent blockers, multivalent therapeutics, and vaccines to boost both humoral and cellular immunity against the virus.This article is categorized under:Therapeutic Approaches and Drug Discovery > Emerging TechnologiesDiagnostic Tools > BiosensingTherapeutic Approaches and Drug Discovery > Nanomedicine for Infectious DiseaseTherapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease

Digital Nucleic Acid Signal Amplification Platform for Highly Sensitive DNA Mutation Analysis.

Digital nucleic acid analysis technology has shown great application potential due to its excellent performance. However, most current digital nucleic acid detection methods are based on PCR or other template amplification strategies. Here, we present an alternative analysis platform based on digital nucleic acid signal amplification in droplets termed dNASA. Using a bead-based controllable extension bridged cascade signal amplification reaction, we achieved an ultralow background, high efficiency, and highly specific nucleic acid signal amplification analysis. As a "proof of concept", we demonstrated the feasibility of the proposed dNASA platform in single-base DNA mutation analysis using artificially synthesized samples. This platform provides innovative ideas for the field of digital nucleic acid analysis.

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms.

In the fight against the worldwide pandemic coronavirus disease 2019 (COVID-19), simple, rapid, and sensitive tools for nucleic acid detection are in urgent need. PCR has been a classic method for nucleic acid detection with high sensitivity and specificity. However, this method still has essential limitations due to the dependence on thermal cycling, which requires costly equipment, professional technicians, and long turnover times. Currently, clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors have been developed as powerful tools for nucleic acid detection. Moreover, the CRISPR method can be performed at physiological temperature, meaning that it is easy to assemble into point-of-care devices. Microfluidic chips hold promises to integrate sample processing and analysis on a chip, reducing the consumption of sample and reagent and increasing the detection throughput. This review provides an overview of recent advances in the development of CRISPR-based biosensing techniques and their perfect combination with microfluidic platforms. New opportunities and challenges for the improvement of specificity and efficiency signal amplification are outlined. Furthermore, their various applications in healthcare, animal husbandry, agriculture, and forestry are discussed.

Visual Detection of Duck Tembusu Virus With CRISPR/Cas13: A Sensitive and Specific Point-of-Care Detection.

Duck tembusu virus (DTMUV), which causes huge economic losses for the poultry industries in Southeast Asia and China, was first identified in 2010. DTMUV disease has become an important disease that endangers the duck industry. A sensitive, accurate, and convenient DTMUV detection method is an important means to reduce the occurrence of the disease. In this study, a CRISPR/Cas13a system was combined with recombinase polymerase amplification to develop a convenient diagnostic method to detect DTMUV. The novel method was based on isothermal detection at 37°C, and the detection was used for visual readout or real-time analysis. The assay was highly sensitive and specific, with a detection limit of 1 copy/μL of the target gene and showed no cross-reactivity with other pathogens. The enhanced Cas13a detection worked well with clinical samples. Overall, a visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a proved to be a powerful tool for detecting DTMUV.

A CRISPR-based nucleic acid detection method for severe fever with thrombocytopenia syndrome virus

ObjectiveFever with thrombocytopenia syndrome virus (SFTS) is a tick-borne infection now known to spread among humans as an aerosol, which has resulted in several outbreaks across Asia over the past decade. As mortality is substantial, it is vital to establish a rapid, on-site nucleic acid detection method for diagnosis. Here we describe such a method for SFTSV (Dabie bandavirus) based on CRISPR-Cas13a. MethodsSpecific recombinase-aided amplification (RAA) primers and CRISPR (cr)RNA nucleic acid detection targets were designed and synthesized for the conserved sequence of the SFTSV genome, and fluorescent CRISPR detection was used to screen for high-sensitivity crRNAs. Colloidal immunochromatography test paper was used to read CRISPR detection results. Sensitivity and specificity were evaluated by running tests on gradient dilutions of SFTSV nucleic acid and the nucleic acids of other pathogens with similar transmission routes or clinical manifestations. ResultsOne crRNA with high detection sensitivity was screened out of 5 crRNAs with conserved sequences from the SFTSV genome. This CRISPR nucleic acid-based detection method was able to detect a single crRNA copy per microliter but not the nucleic acids of similar pathogens. ConclusionThis CRISPR test strip detection method permits rapid, sensitive, and specific diagnosis of SFTS without the need for advanced nucleic acid detection equipment, thus allowing for on-site application.

Recent advances and clinical application in point-of-care testing of SARS-CoV-2.

The novel coronavirus 2019 (COVID‐19) caused by SARS‐CoV‐2 spread rapidly worldwide, posing a severe threat to public life and health. It is significant to realize rapid testing and timely control of epidemic situations under the condition of limited resources. However, laboratory‐based standardized nucleic acid detection methods have a long turnaround time and high cost, so it is urgent to develop convenient methods for detecting COVID‐19. This paper summarizes the point‐of‐care testing (POCT) developed for novel coronavirus from three aspects: nucleic acid extraction, nucleic acid amplification, and detection methods. This paper introduces a commercial real‐time detection system that integrates the abovementioned three steps and the matters needing attention in use. The primary purpose of this review is to provide a reference for emergency response and rapid deployment of COVID‐19 and some other emerging infectious diseases.

CRISPR Approaches for the Diagnosis of Human Diseases.

CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.

Rapid and Specific Detection of Active SARS-CoV-2 With CRISPR/Cas12a.

Rapid and sensitive nucleic acid detection of SARS-CoV-2 has contributed to the clinical diagnosis and control of COVID-19. Although detection of virus genomic RNA (gRNA) has been commonly used in clinical diagnosis, SARS-CoV-2 gRNA detection could not discriminate between active infectious virus with remnant viral RNA. In contrast to genomic RNA, subgenomic RNAs (sgRNAs) are only produced when the virus is actively replicating and transcription, detection of sgRNA could be an indication to evaluate infectivity. CRISPR/Cas-based nucleic acid detection methods have been considered potential diagnostic tools due to their intrinsic sensitivity, specificity and simplicity. In this study, to specifically detect active virus replication, we developed a CRISPR-based active SARS-CoV-2 (CRISPR-actCoV) detection strategy by detecting sgRNAs of SARS-CoV-2. CRISPR-actCoV with CRISPR Cas12a-assisted fluorescence reporter system enables detection of sgRNAs at 10 copies in 35 min with high specificity and can be read out with naked eyes. Further, we performed CRISPR-actCoV mediated sgRNA detection in 30 SARS-CoV-2 potentially infected clinical samples, and 21 samples were SARS-CoV-2 sgRNA positive. A quantitative RT-PCR assay was also performed to detect gRNA of SARS-CoV-2 in parallel. Among the 30 clinical samples, 27 samples were gRNA positive. Taken together, CRISPR-actCoV provides an alternative for rapid and accurate detection of active SARS-CoV-2 and has great significance in better response of coronavirus causing epidemic disease.

The Recent Original Perspectives on Nonculture-Based Bacteria Detection Methods: A Comprehensive Review.

Foodborne diseases that are primarily caused by pathogenic bacteria are of major public health concern globally. One of the key strategies in minimizing and controlling the risk of contamination of food with such pathogens requires establishing effective detection and tracking methods of zoonotic bacteria. Although culture-based pathogen detection methods are developed and widely used by the industry traditionally, nonculture-based zoonotic bacteria detection methods are now more widely investigated and used owing to the recent developments in nucleic acid and immunological-based detection methods. These rapid detection methods provide the opportunity of acquiring real-time test results and high-throughput screening of a large number of samples at a time. One of the key aspects of rapid detection methods is the development of effective sample processing methods as food samples are heterogeneous and highly complex in composition. In this review, variety of sample processing methods, in terms of nonspecific and target-specific sample processing as well as thorough overview of recent developments in nonculture-based bacteria detection methods are presented.

Droplet digital recombinase polymerase amplification (ddRPA) reaction unlocking via picoinjection

Recombinase polymerase amplification (RPA) has been recognized as a promising isothermal amplification method for nucleic acid detection. However, the digital format of RPA is still challenging to implement due to its MgOAc-initiated reaction feature and the inherent non-specific amplification. Here we develop a Picoinjection Aided Digital reaction unLOCKing (PADLOCK) approach utilizing droplet microfluidics to achieve droplet digital RPA (ddRPA) for absolute nucleic acid quantification. By coupling a microfluidic picoinjector with a droplet generator, the reaction initiator MgOAc is dosed into droplets containing MgOAc-deprived RPA master mix for controlled digital reaction unlocking, which completely circumvents premature amplification. The discretization of the targets to a single-molecule level in confined droplets endows absolute quantification of the copy number. Coupled with CRISPR/Cas13a sensing, the ddRPA demonstrates single molecule detection ability within 30 min with significantly enhanced signal-to-noise ratio (S/N ratio>6) and uniform fluorescence signal reporting, facilitating the precise quantification of nucleic acids. Furthermore, the utility of the PADLOCK-CRISPR assay has been validated with 22 clinical samples, which generated results in 100% concordance with qPCR. We believe the coupling of droplet microfluidic technology with digital RPA will pave the way towards ultrasensitive and precise nucleic acid quantification.